Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs¹. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12–q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs^2,3. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy⁵ (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.

Trophoblast invasion of the uterine extracellular matrix, a critical process of human implantation and essential for fetal development, is a striking example of controlled invasiveness. To identify molecules that regulate trophoblast invasion, mRNA signatures of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared using U95A GeneChip microarrays yielding 220 invasion/migration-related genes. In this `invasion cluster', KiSS-1 and its G-protein-coupled receptor KiSS-1R were expressed at higher levels in first trimester trophoblasts than at term of gestation. Receptor and ligand mRNA and protein were localized to the trophoblast compartment. In contrast to KiSS-1, which is only expressed in the villous trophoblast, KiSS-1R was also found in the extravillous trophoblast, suggesting endocrine/paracrine activation mechanisms. The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145), but shorter kisspeptins (Kp) with 10, 13, 14 or 54 amino acid residues may be produced. We identified Kp-10, a dekapeptide derived from the primary translation product, in conditioned medium of first trimester human trophoblast. Kp-10, but not other kisspeptins, increased intracellular Ca²⁺ levels in isolated first trimester trophoblasts. Kp-10 inhibited trophoblast migration in an explant as well as transwell assay without affecting proliferation. Suppressed motility was paralleled with suppressed gelatinolytic activity of isolated trophoblasts. These results identifed Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.

Activation of endothelial cells by lipid oxidation products is a key event in the initiation and progression of the atherosclerotic lesion. Minimally modified low-density lipoprotein (MM-LDL) induces the expression of certain inflammatory molecules such as tissue factor (TF) in endothelial cells. This study examined intracellular signaling pathways leading to TF up-regulation by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a biologically active component of MM-LDL. OxPAPC induced TF activity and protein expression in human umbilical vein endothelial cells (HUVECs). However, OxPAPC neither induced phosphorylation or degradation of IκBα nor DNA binding of nuclear factor-κB (NF-κB). Furthermore, OxPAPC-induced TF expression was not inhibited by overexpression of IκBα. These results strongly indicate that OxPAPC-induced TF expression is independent of the classical NF-κB pathway. However, OxPAPC stimulated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and expression of early growth response factor 1 (EGR-1). Inhibitors of mitogen-activated kinase/ERK (MEK) or protein kinase C (PKC) blocked elevation of both EGR-1 and TF. Furthermore, overexpression of NAB2, a corepressor of EGR-1, inhibited effects of OxPAPC. In addition, OxPAPC induced rapid and reversible elevation of free cytosolic Ca⁺⁺ levels and nuclear factor of activated T cells (NFAT)/DNA binding. Induction of TF expression by OxPAPC was partially inhibited by cyclosporin A, known to block calcineurin, a Ca⁺⁺-dependent phosphatase upstream of NFAT. Treatment of OxPAPC with phospholipase A₂ destroyed its biologic activity and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine was identified as one biologically active component of OxPAPC that induces TF expression. Together, the results demonstrate that OxPAPC induces TF expression in HUVECs through activation of PKC/ERK/EGR-1 and Ca⁺⁺/calcineurin/NFAT pathways rather than by NF-κB–mediated transcription. Thus, oxidized phospholipids may contribute to inflammation by activating pathways alternative to the classical NF-κB pathway.

Changes in intra- and extracellular potassium ion (K⁺) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K⁺ changes is severely limited by the lack of practicable detection methods. We developed K⁺-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K⁺ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K⁺ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K⁺ signals. Hitherto unknown Ca²⁺-triggered, organelle-specific K⁺ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K⁺ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K⁺ assays and open new avenues for live-cell K⁺ imaging.

Adipose triglyceride lipase (ATGL) catalyzes the first step in the hydrolysis of triacylglycerol (TG) generating diacylglycerol and free fatty acids. The enzyme requires the activator protein CGI-58 (or ABHD5) for full enzymatic activity. Defective ATGL function causes a recessively inherited disorder named neutral lipid storage disease that is characterized by systemic TG accumulation and myopathy. In this study, we investigated the functional defects associated with mutations in the ATGL gene that cause neutral lipid storage disease. We show that these mutations lead to the expression of either inactive enzymes localizing to lipid droplets (LDs) or enzymatically active lipases with defective LD binding. Additionally, our studies assign important regulatory functions to the C-terminal part of ATGL. Truncated mutant ATGL variants lacking approximately 220 amino acids of the C-terminal protein region do not localize to LDs. Interestingly, however, these mutants exhibit substantially increased TG hydrolase activity in vitro (up to 20-fold) compared with the wild-type enzyme, indicating that the C-terminal region suppresses enzyme activity. Protein-protein interaction studies revealed an increased binding of truncated ATGL to CGI-58, suggesting that the C-terminal part interferes with CGI-58 interaction and enzyme activation. Compared with the human enzyme, the C-terminal region of mouse ATGL is much less effective in suppressing enzyme activity, implicating species-dependent differences in enzyme regulation. Together, our results demonstrate that the C-terminal region of ATGL is essential for proper localization of the enzyme and suppresses enzyme activity.